Methods for treating or reducing prediposition to breast cancer, pre-menstrual syndrome or symptoms associated with menopause by administration of phyto-estrogen

ABSTRACT

Phyto-estrogen-containing health supplement compositions containing any two or more of Genistein, Daidzein, Formononetin and Biochanin A, or the natural glycosides thereof are administered for treating or reducing predisposition to breast cancer, pre-menstrual syndrome or symptoms of menopause.

This is a continuation of U.S. application Ser. No. 08/338,567, filedJan. 12, 1995 now U.S. Pat. No. 5,830,887, which is a 371 ofPCT/AU93/00230 filed May 19, 1993.

TECHNICAL FIELD

This invention relates to natural products containing phyto-oestrogens,or phyto-oestrogen metabolites, which have various beneficialphysiological effects in man, and which have a variety of uses, such asto promote good health and as a dietary additive, for example.

BACKGROUND ART

The particular product in accordance with the invention is an extract ofcertain plants with the particular purpose of enrichment forphyto-oestrogens, both in their natural state and their closely relatedderivatives and metabolites.

Plants which are used as foodstuffs or medicinal herbs contain a widevariety of chemicals which are assimilated into the body followingingestion. Some of these chemicals are important nutrients for man andanimals (e.g. fats, carbohydrates, proteins, vitamins, minerals) whileothers have none, or little or no known nutritional value. Thephyto-oestrogens hitherto have fallen into this latter category of noknown nutritional value.

There are 3 principal classes of phyto-oestrogens, viz. isoflavones,lignans, and coumestans. The isoflavones are thought to have a broadrange of biological functions in plants, although these are poorlyunderstood. However, two particular functions are recognised—(a) asphyto-alexin or stressor chemicals which are secreted by the plant inresponse to attack by parasites such as insects, fungi, viruses, etc andwhich display activity against these parasites, and (b) chemicals whichencourage colonisation of nitrogen-fixing bacteria on the roots oflegumes. The biological functions in plants of the lignans andcoumestans is not generally understood.

The different types of phyto-oestrogens are as follows.

Type 1 phyto-oestrogens—(isoflavones) Isoflavones appear to be widelydistributed in the plant kingdom and over 700 different isoflavones aredescribed. However, the isoflavones which display oestrogenic activitybelong to a small sub-group and are restricted almost exclusively to theLeguminosae family. The known oestrogenic isoflavones are daidzein,formononetin, genistein and biochanin A. In common human foodstuffs suchas soya, chickpeas, lentils and beans, the total levels of theoestrogenic isoflavones range between about 40 and 300 mg per 100 g dryweight.

In the raw plant material, isoflavones occur principally as glycosides.Following ingestion by man and animals, the glycoside moiety ishydrolysed free by a combination of gastric acid hydrolysis andfermentation by intestinal bacteria. Some of the isoflavones in theaglucone form are absorbed directly and circulate in the blood, whilethe remainder are metabolised by intestinal fermentation to a variety ofcompounds which are also absorbed. The absorbed isoflavones and theirmetabolites appear to undergo little or no further metabolism in thebody, being readily transported in the bloodstream, and ultimately beingexcreted in the urine.

Type 2 phyto-oestrogens (lignans).

Lignans are widely distributed in the plant kingdom. Over one hundredlignans are described and they are reported in common human foodstuffssuch as cereals, fruits and vegetables. Oilseeds such as flax (linseed)have the highest known levels at 20-60 mg/100 g dry weight, whilecereals and legumes have much lower levels at 0.3-0.6 mg/100 g, andvegetables even lower levels at 0.1-0.2 mg/100 g. The most common lignandescribed is metairesinol. Dietary lignans also appear to be metabolisedfairly efficiently within the gut by bacterial fermentation, yieldingmetabolites such as enterodiol and enterolactone which are absorbed intothe bloodstream and excreted in the urine.

Type 3 phyto-oestrogens (coumestans).

Compared to isoflavones and lignans, oestrogenic coumestans appear tohave a relatively restricted distribution in plants and generally occurat much lower levels. Alfalfa, ladino clover and some other fodder cropssuch as barrel medic may have significant levels and have been reportedto cause reproductive dysfunction in grazing animals. In the human diet,the important sources of coumestans are sprouts of soya and alfalfawhere levels up to 7 mg/100 g dry weight are reported. Whole soyabeansand other common foodstuff legumes contain levels of approx. 0.12 mg/100g dry weight and most of that is concentrated in the seed hull whichcommonly is removed in the preparation of human foodstuffs.

Type 4 phyto-oestrogens (oestrogens).

These are compounds closely related to animal oestrogens such asoestrone, oestradiol and oestriol. These have been described in plantssuch as liquorice, apple, French bean, pomegranate and date palm. Littleis known of the metabolism and biological significance of thesechemicals in humans and animals.

The full range of biological effects in animals of these dietaryphyto-oestrogens has received only recent study. A primary effectappears to be associated with their close structural relationship tonaturally-occurring oestrogens which allows the phyto-oestrogens tomimic the effects of the endogenous oestrogens. The known biologicaleffects of phyto-oestrogens can be summarised thus:

In vitro (a) bind to both cytoplasmic and nuclear membrane (Type II)oestrogen receptors on human tissues; (b) strongly compete withoestrogens for oestrogen receptors, but only weakly stimulate thosereceptors; (c) strongly stimulate the production of sex hormone-bindingglobulin (SHBG) from human cells; In vivo (d) weakly oestrogenic inanimals; (e) competitively-inhibit the response of tissue to oestrogens.

The three major types of phyto-oestrogens appear to act at the cellularlevel in a similar manner, that is through interaction with cell surfaceoestrogen receptors. In the body, naturally-occurring oestrogenscirculating in the blood largely exert their activity by interactionwith oestrogen receptors on cell surfaces; such interactions thentriggering a particular biological function of that particular cell.Phyto-oestrogens are able to bind to those oestrogen receptors becausethe structure of these compounds so closely resembles the endogenousoestrogens, but unlike the animal oestrogens, phyto-oestrogens onlyweakly activate the oestrogen receptor.

As a result of phyto-oestrogens and endogenous oestrogens competing forthe oestrogen-binding sites on cells, the more weakly oestrogenicphyto-oestrogens can be considered to have an anti-oestrogenic effect.This phenomenon is known as competitive-inhibition, by which is meantthat the biological effect of an active substance is impaired by thecompetitive binding to a target receptor of a similar but less activecompound.

Thus a primary biological effect of phyto-oestrogens is held to becompetitive inhibition of endogenous oestrogens. However, another moredirect effect is the stimulation of synthesis of SHBG in the liver, asoccurs with orally administered synthetic steroidal oestrogens. Highlevels of dietary phyto-oestrogens are thought to be responsible for thehigher SHBG levels seen in vegetarians and in cultures maintainingtraditional (high legume-containing) diets.

At high levels, dietary phyto-oestrogens can have profound physiologicaleffects. An example of this is sheep and cattle grazing pasturescontaining a high proportion of subterranean clover or red clover whichcan contain levels of phyto-oestrogens as high as 5% of the dry weightof the plant. As a result of the competitively-inhibitory effect of thedietary phyto-oestrogens on endogenous oestrogen function in thehypothalamus, male and female sheep and cows can develop androgenicsymptoms.

Such high dietary levels of phyto-oestrogens, however, are rare. It isfar more common that most animal and human diets contain low to moderatelevels of phyto-oestrogens, and there is growing epidemiologicalevidence that such levels have a beneficial effect on human health.

In most traditional human diets in developing countries, the principalphyto-oestrogens consumed are isoflavones because of the generally highreliance on legumes (also known as pulses) as a source of protein. Thegeneral consumption rates (g/day/person) for legumes for differentregions currently are approximately: Japan (50-90), India (40-80), SouthAmerica (30-70), North Africa (40-50), Central/Southern Africa (20-50)and Southern Mediterranean (30-60). Legumes also are a source of lignansand, to a much lesser extent, coumestans, and the additional cereal andvegetables in the diet would also boost the lignan intake. However, theisoflavone intake in these traditional cultures with high legumeconsumption would typically be much in excess of either lignan orcoumestan intake.

The major types of legumes used in traditional diets include soya,chickpeas, lentils, ground nuts, beans (e.g. broad, haricot, kidney,lima, navy), and grams (bengal, horse and green).

In Western, developed countries, the daily intake of dietaryphyto-oestrogens generally is negligible to low. In Western Europe,North America and Australasia, legumes were a major source of proteinfor the majority of the populations up to the end of the 19th century.From that time, legume consumption has declined significantly, beingreplaced in the diet with protein of animal origin. Average legumeconsumption in these regions currently is between 5-15 g/day/person witha significant proportion of the population ingesting little to nolegumes or other phyto-oestrogen containing foods on a regular basis.Moreover, the types of legumes consumed in these regions (e.g. gardenpeas, French beans) have a typically lower isoflavone content thanlegumes such as soya and chick peas.

Based on typical consumption rates and types of foodstuffs consumed, thetypical phyto-oestrogen intake (mg/day) for different regions can becalculated approximately as

Isoflavones Lignans Coumestans Japan 50-300 2-5 0.5 Australia 2-25 1-50.2

Thus it can be seen that regions which have maintained traditional dietshave a higher average daily intake of phyto-oestrogens, particularlyisoflavones, compared to western countries. People in communities suchas Japan or developing countries with high legume intake excretesubstantially higher phyto-oestrogen metabolites in their urine comparedto people in Western countries. Within the latter, vegetarians alsoexcrete higher phyto-oestrogen metabolite levels than do those consuminga more typical, omnivorous Western diet.

The presence of relatively large amounts of phyto-oestrogen metabolitesin urine serves to highlight their potential biological significance. Ithas been shown that total urinary excretion of isoflavones and theiractive metabolites in people consuming moderate amounts of legumes isgreatly in excess (up to 10,000×) of steroidal oestrogen levels. So thatwhile the oestrogenicity of isoflavones to oestrogen receptors is onlyabout 1% that of endogenous oestrogens, this weaker effect is off-set bythe much higher blood levels of the isoflavones.

It is known that legumes have formed an important part of the human dietover the past 20,000-30,000 years. It therefore follows that humanmetabolism has evolved over at least this period in the presence ofrelatively large levels of dietary phyto-oestrogens, particularlyisoflavones. Given the known biological effects of phyto-oestrogens, italso follows that endogenous oestrogen metabolism and function hasevolved in the face of significant competitive inhibiting effects ofphyto-oestrogens. It has been speculated that the presence ofsignificant dietary levels of phyto-oestrogens in recent human evolutionhas led to a degree of adaption by tissues responsive to reproductivehormones to these dietary components. That is, both the rate ofproduction and/or the function of endogenous oestrogens may be eitherdependent upon or influenced by the presence of phyto-oestrogens in thebody. It follows therefore that a relative deficiency of dietaryphyto-oestrogens could be expected to lead to an imbalance of endogenousoestrogen metabolism.

There is increasing interest in the likely contribution of a relativedeficiency of dietary phyto-oestrogens to the development of theso-called “Western diseases”, especially cancer of the breast, benign(cystic) breast disease, cancer of the uterus, cancer of the prostate,cancer of the bowel, pre-menstrual syndrome, menopausal syndrome, andatherosclerosis. All of these diseases are associated to a greater orlesser extent to oestrogen metabolism, and oestrogen function is eitherknown or is suspected to play a role in their aetiology and/orpathogenesis.

Each of these diseases occurs at much higher incidence in Western,developed countries than it does in developing communities. Moreover, itis thought that in Western communities, the incidences of each haverisen over the past century. It is also generally held, that of all theenvironmental factors likely to be contributing to this phenomenon, dietis the principal factor. Of those dietary components with the potentialto influence the aetiology of oestrogen-related disease, there is agrowing awareness that phyto-oestrogens may have important potential.

The beneficial effects of phyto-oestrogens on human health are thoughtto derive from at least two principal function, those being (i)competitive-inhibition of the function of endogenous oestrogens, and(ii) the stimulation of production of SHBG. SHBG plays an important rolein primates in binding and transporting the reproductive hormones(oestrogens, androgens) in blood so that the availability ofreproductive hormones is regulated to a large degree by SHBG levels.Higher SHBG levels are considered beneficial in leading to a reductionin both blood levels of unbound (and unregulated) reproductive hormonesand metabolic clearance rates of the hormones. Although isoflavones arepotent stimulators of SHBG synthesis, they only weakly bind to SHBG, sothat the increased SHBG levels resulting from the dietary isoflavonesare largely available for binding to endogenous oestrogens.

In terms of directly identifying the beneficial effects ofphyto-oestrogens in amelioration of any or all of the “Westerndiseases”, there are only two examples. In one example, the diets ofwomen, with menopausal syndrome were supplemented with foodstuffs (soya,linseed, red clover) high in phyto-oestrogens, and an alleviation ofmenopausal symptoms to an extent similar to that obtained withreplacement therapy with synthetic oestrogens was achieved; that effectwas ascribed to the phyto-oestrogen content of the supplement. In theother example, legumes such as soya and various pulses have been shownto have a hypocholesterolaemic effect in humans; this effect has notbeen ascribed to phyto-oestrogens, although purified isoflavones do havea hypocholesterolaemic effect in animals with artificially-inducedhypercholesterolaemia.

In summary, it could reasonably be deduced that the inclusion of greaterlevels of foodstuffs high in phyto-oestrogens in the standard diets ofmen and women in developed countries could be expected to redress ageneral imbalance of endogenous reproductive hormone metabolism, therebyreducing the predisposition of those communities to the above diseases.While there are various types of phyto-oestrogens which may be suitableto this end, the large discrepancy in isoflavone consumption betweencommunities with Western and traditional diets suggest that foodstuffswith high isoflavone content are of prime interest.

However it is unrealistic to expect that public education programmeswould readily convert communities in developed countries from a dietwhere the protein content is predominantly animal-derived, to one wherethe protein is predominantly legume-derived. Moreover, the legumes whichare commonly consumed in developed countries are relatively poor sourcesof phyto-oestrogens and the general acceptance in the community of lesswell-known legumes with higher phyto-oestrogen content would benecessarily a slow process. Also, the highly variable levels ofphyto-oestrogens in foodstuffs relating to plant strain type, degree ofplant maturity, and climatic and other environmental conditions suggeststhat the supply of an assured amount of phyto-oestrogens through the useof whole foodstuffs may be difficult.

An alternative strategy is to make available either (i) phyto-oestrogensin a purified form, or (ii) foodstuffs which are enriched forphyto-oestrogens. In this way, the phyto-oestrogen could be added to thediet in a convenient form as a supplement without requiring anysubstantive change to the diet.

DISCLOSURE OF INVENTION

The present invention concerns a health supplement specifically enrichedfor isoflavones selected from genistein, daidzein, formononetin andbiochanin A, or their natural glycoside form, or their analogues, insufficient amounts to improve the health of a human.

Preferably the supplement contains an excipient, a diluent, a carrier orthe like, or else the supplement is mixed with food or can be consumeddirectly. It is also preferred that foodstuffs, are readily available,have no known toxic components, and are rich sources of isoflavones;such foodstuffs preferably being red clover or soya. It is alsopreferred that the ratio of genistein and/or it methylated derivativebiochanin A to daidzein and/or its methylated derivative formononetin isbetween 1:2 to 2:1. Other plant components with oestrogenic activityincluding lignans, coumestans and flavones may also be present in theextract, but it is held that these are of secondary importance to thepredominant isoflavones. The term phyto-oestrogens is used hereafter toindicate a predominance of isoflavones with lesser amounts of lignans,coumestans and flavones.

The invention also concerns a method of improving the health of a humanby administering to the human a sufficient amount of phyto-oestrogen.Ideally, the phyto-oestrogen is administered regularly on a daily basisover a sufficient period such as at least a month. The health conditionswhich may be prevented or ameliorated include cancer of the breast,cancer of the prostate, cancer of the uterus, cancer of the bowel,benign (or cystic) breast disease, pre-menstrual syndrome (also known aspre-menstrual tension), or adverse symptoms associated with menopause inwomen. The method and supplement in accordance with the invention alsoimproves the health of a human having elevated levels of bloodcholesterol. The product also is useful in avoiding or amelioratingcancer in persons. The symptoms produced by these conditions and thegeneral well-being is also improved by the use of these supplements

The phyto-oestrogen in accordance with the invention may be obtainedfrom a number of different sources. Preferably the phyto-oestrogens areextracted from a clover such as red clover or subterranean clover orfrom soya which contain high levels of phyto-oestrogens. However, anysource rich in phyto-oestrogens may be used instead, if desired.

Various different isoflavones have been identified from these sources -they are principally genistein, biochanin A, daidzein, formononetin andglycitein. In plants these compounds occur principally in a glycosideform bound to sugars such as glucose, with smaller amounts present asthe aglucone forms. The formulae of the isoflavones are:

The structure of biochanin A is the same as for genistein but with a4′-methoxy group, and similarly formononetin has the same structure asdaidzein, but with a 4′-methoxy group.

Following ingestion by humans, the glycosidic isoflavones are hydrolysedto the aglucone form and biochanin A and formononetin are demethylatedby bacterial fermentation to genistein and daidzein respectively. Asmall proportion of these free isoflavones are absorbed directly fromthe bowel and circulate in the blood. The bulk of the isoflavones,however, remain in the bowel and undergo fermentation to form variousmetabolites which also are absorbed into the bloodstream. The principalmetabolites which have been identified are equol andO-desmethylangolensin.

In vitro and in vivo studies have indicated that genistein, biochanin A,equol, daidzein, formononetin all have oestrogenic activity indescending order. O-desmethylangolensin is only very weakly oestrogenicand glycitein is non-oestrogenic.

In animal and in vitro studies, genistein has been shown to have greateroestrogenic/anti-oestrogenic activity and SHBG-stimulating capacity thanthe other isoflavones or their metabolites (approximately 10 times thatof daidzein and formononetin). However, the full range of biologicaleffects of the different isoflavones have yet to be fully determined,and in particular their relative efficacies in the different biologicaleffects such as oestrogenicity, hypocholesterolaemia, anti-angiogenesis,anti-oxidation, anti-carcinogenesis for example are not yet fully known.

It is thought that because the methyl forms (biochanin A andformononetin) ultimately are largely demethylated to their principals,genistein and daidzein, with improved biological efficacy, then it isunimportant whether the isoflavones are present in the claimed productin the methylated or demethylated forms.

Given that the relative biological importance of the two isoflavonegroups (being genistein and daidzein) to human health remains unclear,and that each might indeed have different importance, plus the fact thatboth isoflavones are present in the diet in approximately equalproportions, then it is prudent that both isoflavones be present in theclaimed product in approximately equal proportions.

Any leguminous plants such as detailed here could be used as sources ofphyto-oestrogens (principally isoflavones with lesser amounts of lignansand coumestans): Indian liquorice (Abrus precatorius); various speciesof Acacia spp. including, A. aneura, A. cibaria, A. longifolia, and A.oswaldii; ground nut (Apio tuberosa); ground pea (Arachis hypogea); milkvetch (Astragalus edulis); marama bean (Bauhinia esculenta); sword bean(Cajanus cajan indicus); jack bean (Canavalia ensiformis); sword bean(Canavalia gladiata); seaside sword bean (Canavalia rosea); variousCassia spp. including C. floribunda, C laevigata, and C. occidentalis;carobbean (Ceratonia siliqua); chick pea (Cicer arietinum); yebnut(Cordeauxia edulis); various Crotalaria spp. including C. laburnifolia,and C. pallida; cluster bean (Cyamopsis psoralioides); tallow tree(Detariaum senegalense); sword bean (Entada scandens); balu (Erythrinaedulis); soyabean (Clycine max); inga (Ingaedulis); Polynesian chestnut(Inocarpus fagifer); hyacinth bean (Lablab purpureus); grass pea orIndian vetch (Lathyrus sativus); cyprus vetch (Lathyrus ochrus); lentil(Lens culinaris); jumping bean (Leucaenal eucocephala); various Lupinusspp. including L. albus, L. luteus, L. angustifolium, L. mutabilis, andL. cosentinii; ground bean (Macotylma geocarpa); horse gram (Macrotylomauniflorum); alfalfa (Medicago sativa); velvet bean (Mucuna pruriens);yam beans (Pachyrhyzuz erosus, P. tuberosus); African locust bean(Parkia clappertoniana); Parkia speciosa; oil bean tree (Pentaclethramacrophylla); various Phaseolus spp. including P. acutifolius, P.vulgaris, P. luntus, P. coccineus, P. adenathus, P. angulris, P. aureus,P. calcaratus, P. mungo, and P. polystachyus; garden pea (Pisumsativum); djenko bean (Pithecolobium lobatum); mesquite (variousProsopis spp.); goa bean (Psophocarpus scandens, P. tetragonolobus);various Psoralea spp.; Sesbania bispinosa; yam bean (Sphenostylisstenocarpa); tamarind (Tamarindus indica); fenugreek (Trigonellafoenum-graecum); vetches (various Vivia spp. including V. sativa, V.atropurpurea, V. ervilia, and V. monantha); broad bean (Vicia faba);black grani (Vigna mungo); various Vigna spp. including V. radiata, V.aconitifolia, V. adanatha, V. angularus, V. tribolata, V. umbelata, andV. unguiculata; and, earth pea (Voandzeia subterranea).

The ideal sources of phyto-oestrogens for preparation of a supplement inaccordance with the invention are preferably those which (i) are readilyavailable, (ii) are relatively inexpensive, (iii) are readily andeconomically processed so as to yield the extract, (iv) have a highisoflavone content so as to provide high yields, and (v) have no knowntoxic components requiring selective removal or inactivation.

Certain clovers, such as red clover (T. pratense) and subterraneanclover (T. subterranean) are the preferred sources. On a dry weightbasis, these clovers contain the highest amounts of oestrogenicisoflavones of all legumes tested to date with levels of 3-5 g % (T.subterranean) and 1-3 g % (T. pratense). In comparison, soya flour has alevel of 0.15-0.30 g %, lentils (0.08-0.12 g %), chick peas (0.07-0.13 g%), and garden peas (0.02-0.03 g %). Thus it can be seen that cloverscontain approximately at least 10-30 times by weight the isoflavonecontent of other commonly available, human leguminous foodstuffs meaningthat for manufacturing purposes, the yield of isoflavones per unitweight of plant material is many times greater from clover than fromother legumes.

Red clover and subterranean clover also are common fodder crops and arereadily grown and are widely available. Clovers also are comparativelycheaper ($200/tonne) than crops such as soya and lentils ($500/tonne).

With clovers, the isoflavones are recovered from the leaf rather thanfrom the seed in the case of soya, beans, nuts and grams. This providesa substantially higher yield of isoflavones per unit area of pasture forclovers compared to other legumes because of the greater leaf mattercompared to seed matter recovered per plant.

Clovers also have an extended growing season, and faster growth ratescompared to those legumes such as soya, lentils or chick peas where theseed is the end-product. Clover can be cropped for its leaf contentrepeatedly over a single growing season. An additional benefit of thisis that as phyto-alexins, the isoflavone content increases in responseto the stress of cropping.

Thus it can be seen that in clovers versus other legumes provide acombination of (a) higher isoflavone content per dry weight of plant,(b) a higher yield of dry matter containing isoflavones per plant, and(c) a higher yield of dry matter per hectare.

An additional feature of clovers is that there are wide varieties ofcultivars with widely differing isoflavone levels and types. This allowsblending of different cultivars to achieve the desired ratio of thedifferent isoflavones, although it is equally possible to use a singlecultivar which provides the desired ratio.

Other legumes such as soyabean flour may be used for enrichment ofphyto-oestrogens but the substantially poorer (approx. 10%) yield ofisoflavones compared to clovers means that the manufacturing costs aresubstantially greater and there is substantially greater amounts ofwaste products which requires disposal or further treatment for re-useas a foodstuff. An alternative, however, to the use of whole soya forthis purpose, is to use the hull, and hypocotyl (or germ) of the wholesoyabean. The hull and hypocotyl represent only a small proportion byweight (8% and 2% respectively) of the intact bean. However, thecoumestrol content of soya is concentrated in the hull, and the daidzeincontent of soya is concentrated in the hypocotyl. The two cotyledonswhich comprise the bulk of the soyabean (90% by weight) contain the bulkof the genistein content of soya. During standard processing ofsoyabeans, the hulls being a fibrous component with little or noperceived nutritional value normally are separated and removed byphysical means. The hypocotyls become separated following the splittingof the cotyledons, and while these currently generally are notdeliberately isolated, they may be separated and isolated by passing thedisturbed soyabeans over a sieve of sufficient pore size to selectivelyremove the small hypocotyl. The hypocotyl contains approx. 1.0-1.5 g %isoflavones (95% daidzein, 5% genistein). The raw hypocotyl and hullmaterial can be ground or milled to produce, for example, a dry powderor flour which then could be either blended or used separately as adietary supplement in a variety of ways including, for example, as apowder, in a liquid form, in a granulated form, in a tablet orencapsulated form, or added to other prepared foodstuffs. Alternatively,it could be further processed to yield an enriched extract ofphyto-oestrogens. Either or both of these materials also could be addedto other leguminous material such as clover to provide the invention.

In plants, the oestrogenic isoflavones are restricted principally to theleaf, fruit and root; the stem and petiole contain very little. Withsoya and other common human legume foodstuff crops, the leaves arerarely regarded as foodstuff, indeed with these crops, the plantsnormally are allowed to die and dry out before the seed crop isharvested. Nevertheless, the fresh leaves of these crops could beregarded as a source of phyto-oestrogens for the invention although themuch lower isoflavone content of the leaves of these crops compared toclovers, plus their generally slow growth compared to clovers, suggeststhat they would not be a preferred source of large-scale isoflavoneenrichment.

To provide a similar amount of isoflavone to that contained in mosttraditional legume-rich diets (50-100 mg oestrogenic isoflavones/day)would require an average daily consumption of 3-6 g dry weight or 15-30g wet weight of specially selected cultivars of clover with particularlyhigh isoflavone levels. Clover grasses generally are not eaten byhumans, except to a limited extent as sprouts of some of the pleasantertasting varieties. Isoflavones are intensely astringent and areresponsible in large part for the bitter taste of legumes. Thus thetypes of bean sprouts, clover sprouts and alfalfa sprouts generallyavailable have been selected on the basis of cultivar and of age forpleasant taste, and in so doing inadvertently have been selected for lowisoflavone content. Of the sprouts currently available in Westerncountries for human consumption, between approx. 100-250 g would need tobe consumed daily to provide a dosage of 50-100 mg isoflavones.Certainly clovers and other legume sprouts are not generally eaten insuch sufficient quantities by humans to obtain the advantages of thepresent invention.

The invention also concerns formulations containing the phyto-oestrogensdiscussed above together with a dietary suitable excipient, diluent,carrier, or with a food. Ideally the formulation is in the form of apill, tablet, capsule, or similar dosage form

The formulations may be a variety of kinds, such as nutritionalsupplements, pharmaceutical preparations, vitamin supplements, foodadditives or foods supplemented with the specified activephyto-oestrogens of the invention, liquid or solid preparations,including drinks, sterile injectable solutions, tablets, coated tablets,capsules, powders, drops, suspensions, or syrups, ointments, lotions,creams, pastes, gels, or the like. The formulations may be in convenientdosage forms, and may also include other active ingredients, and/or maycontain conventional excipients, carriers and diluents. The inclusion ofthe subject phyto-oestrogens in herbal remedies and treatments is also apreferred part of the invention.

The invention is also directed to the amelioration, prevention, or ofvarious conditions responsive to treatment with the phyto-oestrogensubstances of the invention. The preferred amounts to be administered tothe human fall within 20-200 mg on a daily basis. More preferably thedosage is from 50-150 mg on a daily basis, and most preferably at adosage of about 100 mg. If desired greater dosages can be administeredfor therapeutic reasons. In contrast to prior practices such highdosages were not possible. For example, dosages of up to or greater than1000 mg may be suitable to treat some conditions. In order to obtain thebenefits of the invention, the treatment with the isoflavones shouldcontinue for a considerable period, ideally for at least a month, andideally continuously for the whole period for which the healthimprovement advantages should accrue.

The product according to the present invention yields a constant andaccurately known amount of isoflavones. The product is also ideally anatural product, which has advantages for consumer acceptance, and inaccordance with the supposed theory behind the invention may verypossibly be one of the main causes for its beneficial effects. Wholelegumes have a widely variable isoflavone content due to two maincauses: the type of legume and the environmental effect. The type oflegume typically has a wide range of isoflavone content. The miligram ofisoflavone per hundred gram of whole foodstuff (dry weight) is given inthe following table:

Soya Products Whole Soya 150-300 Soya Milk  25-40 (mg per 200 ml) Tofu 55-95 Lentils  80-120 Chickpeas  70-130 Broad beans  15-20 Garden peas 15-25

Thus common leguminous foodstuffs consumed in Western countries (broadbeans, garden peas etc) have relatively low oestrogenic isoflavonecontent and exceptionally large amounts of these would need to beconsumed daily to approximate those isoflavone levels consumed intraditional diets. Most Western cultures do not traditionally eatlegumes with high isoflavone contents, and those soya products (milk,tofu etc) which are becoming increasingly popular in Western countries,also have relatively low isoflavone levels compared to whole soya,indicating that relatively large amounts of these would need to beconsumed on a regular basis to deliver the required isoflavone levels.

The enviromental effect arises because the isoflavone levels in anyspecies of plant depend greatly on the age of the plant, the climaticconditions where it is grown, the fertiliser and so forth. Thereforeconstant and consistant dosage is very difficult with ordinary wholefoodstuffs. The accurately determined quality and quantity of the activeisoflavones in the product, and its easy consumability when comparedwith the almost impossible task of eating huge amounts of oftenpractically inedible foods, is therefore an import feature of theinvention for preventing and helping in overcoming various healthproblems.

Among the various health problems, the treatment or prevention of highblood cholesterol levels, and the treatment of PMS and menopausalsymptoms is especially important. The product of the invention modulatesthe production and/or function of endogenous sex hormones in humans tomodify or produce health improving effects, including the following: (i)lowered levels of various blood lipoproteins including, for instance,low-density and very-low-density cholesterol leading to reduced risk ofdevelopment of atherosclerosis; (ii) reduced risk of development ofcancer of the prostate; (iii) reduced risk of cancer of the breast; (iv)reduced risk of development of cancer of the uterus; (v) reduced risk ofdevelopment of cancer of the large bowel; (vi) reduced risk ofdevelopment of the syndrome in women commonly referred to pre-menstrualsyndrome (PMS), which includes pre-menstrual tension (PMT); (vii)reduced risk of development of many untoward symptoms (including dryvagina, peripheral flushing, depression etc) commonly associated inwomen with menopause; and for treating benign breast disease in women(benign or cystic breast disease associated with non-malignant swellingand tenderness of breast tissue). The invention therefore is directed toa method for the prophylaxis or treatment of a human, to combatconditions associated with phyto-oestrogen deficiency, which comprisesadministering to the human an effective amount of phyto-oestrogenprincipally isoflavone but which might also include relatively smalleramounts of lignans and coumestans, ideally in a concentrated form,wherein the isoflavones include genistein, and/or biochanin A, and/ordaidzein, and/or formononetin.

Cancer of the breast generally is considered to be associated withoestrogenic dysfunction. Breast cancer cells may display more oestrogenreceptors than normal breast cells and stimulation of these receptors byendogenous oestrogens is thought to be a prime source of stimulation oftheir malignant growth. Currently synthetic anti-oestrogens are beingused to prevent or treat the growth of malignant breast cells.Isoflavones are potent anti-oestrogens that could be expected to helpprevent or to successfully treat breast cancer. It has been reportedthat the risk of breast cancer in western societies is indirectlyproportional to the level of phyto-oestrogens in the diet and to theamounts of phyto-oestrogen metabolites excreted in the urine.

Cancer of the prostate generally is considered to be associated with sexhormone dysfunction and the growth of prostatic cancer cells isinfluenced by oestrogens and androgens. The incidence of prostaticcancer is low in communities with high legume intake and, conversely, ishigh in Western socieites. Phyto-oestrogens are though to protect fromdevelopment of prostatic cancer. One mechanism may be the effect ofphyto-oestrogens on lowering the proportion of unbound:boundreproductive hormones in the blood. However, there is other evidence tosuggest that phyti-oestrogens, particularly isoflavones, can have adirect influence on certain cellular enzymes within prostatic cells.

Pre-menstrual syndrome has uncertain aetiology and pathogenesis,although most certainly is associated with reproductive hormonedysfunction. It also is a syndrome which has reportedly lower incidencein communities maintaining traditional high-legume diets. It is proposedthat phyto-oestrogens will alleviate this condition by restoring balanceto oestrogen metabolism.

Menopausal syndrome is associated with changes in the oestrogen profilein the body with advancing age. Adverse clinical symptoms may be treatedwith oestrogen replacement therapy. There is evidence that foodstuffshigh in phyto-oestrogens are a suitable alternative to synthetichormones in this respect, producing alleviation of adverse clinicalsymptoms. Again, it is proposed that phyto-oestrogens will function byrestoring balance to oestrogen metabolism.

Benign (or cystic) breast disease has unknown aetiology. However, itsassociation in women with certain stages of the menstrual cycle isstrongly suggestive of oestrogen dysfunction. There currently is nosuccessful treatment of this condition. Phyto-oestrogens are proposed tosuccessfully treat this condition by restoring balance to oestrogenmetabolism. Atherosclerosis is associated with cholesterol metabolismwhich in turn is associated closely with oestrogen metabolism. Thegenerally higher incidence of atherosclerosis in young men versus youngwomen, the rising incidence in women following menopause, and the lowerincidence in post-menopausal women receiving oestrogen replacementtherapy, all point to the moderating influence of oestrogens oncholesterol metabolism. A prime effect of oestrogens on cholesterolmetabolism is stimulation of the liver to process cholesterol,particularly the highly atherogenic low-density lipoproteins and verylow-density lipoproteins, into bile salts. It is proposed thatphyto-oestrogens have an important hypocholesterolaemic effect inhumans. There may be a variety of mechanisms involved, but one may bethe stimulation by phyto-oestrogens of cholesterol catabolism by theliver.

MODES FOR CARRYING OUT THE INVENTION

The invention is now described with reference to various examples.

EXAMPLE 1 Preparation of Red Clover Product

Tablets were prepared using red clover in accordance with the followingprocedure. The raw plant material is harvested and dried; such dryingbeing either sun-drying or from applied heat The dried product is thenpreferably chaffed, before the following extraction step, although thiscan be omitted if desired.

The dried material is extracted in an aqueous: organic solvent mix. Theaqueous phase is required to extract the water-soluble glycoside form ofisoflavones, while the organic solvent is required to solubilise thewater-insoluble aglycone form. The organic solvent can be eitheralcohol, chloroform, acetone or ethyl acetate. The ratio of solvent inthe water can be between 0.1% and 99.9%. The preferred method is to use60% alcohol in water.

The isoflavones are extracted by exposing the plant material to thewater:solvent mix. The exposure time in general terms is indirectlyproportional to the temperature of the mixture. The temperature of themix can range between ambient temperature and boiling temperature. Theexposure time can be between 1 hour and 4 weeks or even longer. It hasbeen determined that the adequate times for maximal recovery ofisoflavones are 2 weeks at 50° C. and 24 hours at 90° C. The supernatantis separated from the undissolved plant material and the organic solventremoved by distillation. The aqueous supernatant then is concentrated,typically by distillation.

Additional processing steps can be used, if desired, to convert theextracted natural product to capsule, tablet, or other convenient formfor ingestion, using normal techniques for doing this. Otherwise theproduct can be packaged as a convenient food additive.

EXAMPLE 2 Preparation of Soya Hypocotyl Product

Soyabeans were heated in dry air so that the hull became brittle. Thebeans then were processed through a tumble mill which removed the hulland split the bean the two cotyledons and the small-sized hypocotylwhich separated from each other. The light-weight hulls then wereremoved by an air stream. The small-sized hypocotyls were separated fromthe larger cotyledons by sieving through a steel wire mesh withapertures of 1 mm×1 mm. This yielded approximately 87% purity ofhypocotyls with 13% contamination by small cotyledon chips.

Normal soybean processing steps isolate the hulls and then these arediscarded or processed separately for use in human and animalfoodstuffs. The hypocotyls normally are not separated and are processedalong with the cotyledons. However, a small number of soybean processorsare separating hypocotyls by the above methods in order to reduce theastringent taste of soyflour for human consumption, and currently thesehypocotyls are either discarded or processed to flour for use in animalfeed.

EXAMPLE 3 Effect of Administering Red Clover Extract to Humans

Seven normal individuals were studied for the comparative effects of redclover extract and whole legumes on blood cholesterol levels. All theindividuals were consuming a standard Western diet with minimal levelsof legumes.

Three men consumed between 100-150 g haricot or navy beans daily for 3weeks as a supplement to their normal diet. This yielded an approximatedaily isoflavone dosage or between 60-100 mg.

Four other individuals (3 men, 1 woman) consumed 5 g of red cloverextract containing 100 mg isoflavones daily for 3 weeks.

Total serum cholesterol levels were determined immediately before andimmediately following the challenge.

Pre-treatment Post-treatment % change Beans only Patient 1 5.77 5.46−5.4 Patient 2 6.24 6.12 −1.9 Patient 3 7.45 8.51 +14.3 Red cloverextract Patient 5 6.53 5.90 −9.6 Patient 6 7.43 6.63 −10.8 Patient 76.33 5.50 −13.1 Patient 8 6.98 7.28 +4.3

The red clover extract had a significantly (P<0.05) greaterhypocholesterolaemic effect than did the whole beans.

Neither of the treatments produced any untoward side effects, althoughthe whole bean eaters reported greater difficulty with compliance oftreatment than did those taking the red clover extract.

EXAMPLE 4 Effect of Administering Soy Hypocotyls to Humans

Fifteen volunteers (8 women, 7 men) were given 5 g of soy hypocotylcontaining (45 mg daidzein and 5 mg genistein) daily for 2 months. Thehypocotyl was consumed as a powder added to the diet.

The effects on cholesterol levels are shown in the following table. Theindividuals are grouped according to their pre-treatment cholesterollevels (high, medium, low).

Range (mean) unmol/L n Pre-treatment Post-treatment Group 1 6 6.3-8.4(7.1) 5.4-6.5 (6.1) Group 2 6 5.0-6.2 (5.5) 4.7-5.9 (5.1) Group 3 33.3-4.7 (4.2) 3.4-4.6 (4.1)

The results show a significant fall in total cholesterol levels in thoseindividuals with cholesterol levels considered to be at the upper end ofthe normal range.

In addition, 1 woman reported substantial amelioration of her benignbreast disease problem associated with mid-cycle swelling andtenderness, and another woman reported regularisation of her menstrualcycle and reduced menstrual bleeding. Both of these effects wereregarded as beneficial.

No other side-effects were reported as a result of the treatment.

What is claimed is:
 1. A method for treating or reducing thepredisposition to at least one condition selected from the groupconsisting of pre-menstrual syndrome or symptoms associated withmenopause, said method comprising administering to a subject having saidat least one condition a therapeutically effective amount of aphytoestrogen-containing health supplement composition, saidphytoestrogen being extracted from soya or clover and comprising any twoor more members of the group consisting of Genistein, Daidzein,Biochanin A, Formononetin or the natural glycosides of any of saidphyto-estrogens.
 2. The method according to claim 1, wherein saidphytoestrogen is extracted from soy.
 3. The method according to claim 1,wherein said phytoestrogen consists essentially of a) genistein and b)daidzein components, wherein component a) optionally contains biochaninA, and component b) optionally contains formononetin, and the ratio ofa):b) is about 1:2 to 2:1.
 4. The method according to claim 1, whereinthe phytoestrogen is administered in an amount of from about 20 mg to200 mg per day.
 5. The method according to claim 1, wherein thephytoestrogen is administered in an amount of from about 50 mg to 150 mgper day.
 6. The method according to claim 1 whereby the phytoestrogen isadministered at least daily, over a period of at least a month.
 7. Amethod according to claim 1, wherein said composition is administeredfor treating or reducing the predisposition to pre-menstrual syndrome.8. A method for treating or reducing the predisposition to symptomsassociated with menopause, said method comprising administering to asubject having said symptoms a therapeutically effective amount of aphytoestrogen-containing health supplement composition, saidphytoestrogen being extracted from soya or clover and comprising any twoor more members of the group consisting of Genistein, Daidzein,Biochanin A, Formononetin or the natural glycosides of any of saidphytoestrogens.
 9. The method according to claim 8, wherein saidphytoestrogen is extracted from soy.
 10. The method according to claim8, wherein said phytoestrogen consists essentially of a) genistein andb) daidzein components, wherein component a) optionally containsbiochanin A, and component b) optionally contains formononetin, and theratio of a):b) is about 1:2 to 2:1.
 11. The method according to claim 8,wherein the phytoestrogen is administered in an amount of from about 20mg to 200 mg per day.
 12. The method according to claim 8, wherein thephytoestrogen is administered in an amount of from about 50 mg to 150 mgper day.
 13. The method according to claim 8 whereby the phytoestrogenis administered at least daily, over a period of at least a month.
 14. Amethod of preventing or ameliorating premenstrual syndrome or symptomsassociated with menopause in a woman, said method comprisingadministering to the woman an effective amount of a naturally occurring,extracted isoflavone which exhibits estrogenic activity, wherein saidisoflavone is selected from the group consisting of genistein, daidzein,biochanin A, formonoetin, O-desmethylangolensin, and equol, wherein saidadministration of the isoflavone increases excretion of phyto-estrogens.15. The method of claim 14 which comprises administering at least twoisoflavones.
 16. A method for treating or reducing the predisposition toat least one condition selected from the group consisting ofpre-menstrual syndrome or symptoms associated with menopause, saidmethod comprising administering to a subject having said at least onecondition or predisposed to said at least one condition a healthsupplement composition containing a therapeutically effective amount ofa purified phytoestrogen, said phytoestrogen being extracted from soyaor clover and comprising any two or more members of the group consistingof Genistein, Daidzein, Biochanin A, Formononetin or the naturalglycosides of any of said phytoestrogens.
 17. The method according toclaim 16, wherein said composition is administered for treating orreducing the predisposition to symptoms associated with menopause.
 18. Amethod for treating or reducing the predisposition to at least onecondition selected from the group consisting of pre-menstrual syndromeor symptoms associated with menopause, said method comprisingadministering to a subject having said at least one condition orpredisposed to said at least one condition a health supplementcomposition containing a therapeutically effective amount of aconcentrated phytoestrogen, said phytoestrogen being extracted from soyaor clover and comprising any two or more members of the group consistingof Genistein, Daidzein, Biochanin A, Formononetin or the naturalglycosides of any of said phytoestrogens.
 19. The method according toclaim 18, wherein said composition is administered for treating orreducing the predisposition to symptoms associated with menopause.
 20. Amethod of preventing or ameliorating premenstrual syndrome or symptomsassociated with menopause in a woman, said method comprising,administering to the woman an effective amount of a purified naturallyoccurring extracted isoflavone which exhibits estrogenic activity,wherein said isoflavone is selected from the group consisting ofgenistein, daidzein, biochanin A, formononetin, O-desmethylangolensin,and equol, wherein said administration of the isoflavone increasesexcretion of phyto-oestrogens.
 21. The method according to claim 20,wherein said composition is administered for treating or reducing thepredisposition to symptoms associated with menopause.
 22. A method ofpreventing or ameliorating premenstrual syndrome or symptoms associatedwith menopause in a woman, said method comprising, administering to thewoman an effective amount of a concentrated naturally occurringextracted isoflavone which exhibits estrogenic activity, wherein saidisoflavone is selected from the group consisting of genistein, daidzein,biochanin A, formononetin, O-desmethylangolensin, and equol, whereinsaid administration of the isoflavone increases excretion ofphyto-oestrogens.
 23. The method according to claim 22, wherein saidcomposition is administered for treating or reducing the predispositionto symptoms associated with menopause.